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Development of a radioimmunoassay for gastric inhibitory polypeptide Kuzio, Maryanne Daisy 1974

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DEVELOPMENT OF A RADIOIMMUNOASSAY FOR GASTRIC INHIBITORY POLYPEPTIDE by MARYANNE DAISY KUZIO B.Sd., University of Alberta, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1974 i i In presenting this thesis in partial fulfilment of the require^ ments for an advanced degree at the University of Br i t ish Columbia, I agree that the Library shall make i t freely ava i l -able for reference and study, I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his represent-atives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Physiology The University of Brit ish Columbia Vancouver 8, Canada February, 1974 ABSTRACT With the isolation, purification and sequencing of Gastric Inhibitory Polypeptide (GIP), interest turned towards its physiological significance. In order for this to be properly evaluated a means had to be developed for measuring its concentration in serum and tissues. A radioimmunoassay was the method of choice due to the sensitivity, specificity, accuracy and precision. This thesis describes the steps involved in the development of a radioimmunoassay. Specific and sensitive antiserum had to be produced. 125 A method had to be standardized for obtaining a high purity - I labelled GIP. With the attainment of these two goals the assay environment had to be evaluated to yield an optimum incubation volume, Trasylol concentration, plasma concentration, pH and incubation timing. Since the antibody/antigen complexes do not spontaneously precipitate, a method of separation was decided upon which yielded efficient separation coupled with technical practicality. Analysis of data was evaluated and the most appropriate means of correcting for non specific binding was determined. Once the assay was established it had to be investigated qualitatively and quantitatively. First the specificity of the antiserum was tested against other gastrointestinal polypeptides. A serum at various dilutions was measured to compare unknown and standard GIP reactivity with antibody. A quality control chart utilizing 10% CCK-PZN was set up. The fasting normal range for GIP was determined. The assay was then utilized in a series of preliminary studies i v t o e v a l u a t e t h e GIP r e s p o n s e t o f e e d i n g and o r a l g l u c o s e t o l e r a n c e t e s t s . I n i t i a l e v i d e n c e f o r the i n s u l i n o t r o p i c a c t i o n o f GIP was p r e s e n t e d . I n d i c a t e d a l s o was an i m p l i c a t i o n o f GIP i n v o l v e m e n t i n v a r i o u s p a t h o l o g i c a l c o n d i t i o n s . TABLE OF CONTENTS PAGE ABSTRACT , i i i LIST OF TABLES , , > • v i i LIST OF FIGURES , • • • v i l i ACKNOWLEDGEMENTS , . , . . x INTRODUCTION , . . . • • 1 METHODS PRODUCTION OF ANTISERA , , 7 I. IMMUNIZATION SCHEDULE , . 7 I I . VEHICLE OF INJECTION , . , 10 I I I . INJECTION ROUTE AND VOLUME 10 IV. CONJUGATION , , , , . 12 V. BLEEDING AND TREATMENT OF ANTISERA . . . 13 LABELLING OF GIP 15 STANDARDS , , , , 22 CONDITIONS OF ASSAY , , . . , , 22 I. DILUENT! BUFFER!' 22 I I . REACTION VOLUME 24 I I I . ASSAY TUBES 27 IV. INCUBATION TIME 27 V. ASSAY PROCEDURE 30 SEPARATION PROCEDURE 31 TYPES OF ASSAYS . . 35 I. DAMAGE ASSAY 35 I I . DILUTION ASSAY 35 I I I . SENSITIVITY ASSAY 37 IV. STANDARD CURVES . 37 SOURCE OF HORMONE PREPARATIONS 41 ANALYSIS OF DATA , 41 RESULTS I. STANDARD CURVE IMPROVEMENTS , . . . . , , . . , « 45 I I . SPECIFICITY OF ANTISERA , . , 45 i v i PAGE I I I . SENSITIVITY, ACCURACY AND PRECISION 45 IV. PLASMA DILUTION ASSAY , . 53 V. TEN PERCENT CCK-PZN ASSAY 57 V I . NORMAL FASTING GIP SERUM VALUES AND RANGES , , , 57 V I I . POST PRANDIAL RELEASE OF GIP , , . , 60 V I I I . GIP SERUM LEVELS WITH ORAL GLUCOSE TOLERANCE TESTS 60 DISCUSSION 67 BIBLIOGRAPHY 87 LIST OF TABLES TABLE PAGE 1. Immunization Schedule for the Induction of Antibodies to GIP in Rabbits 8 2. Immunization Schedule for the Induction of Antibodies to GIP in Guinea Pigs , 9 3. Immunization Schedule for the Induction of Antibodies to a New Series of Animals (7 Guinea Pigs) 11 4. Cummulative Distribution Determination of Normal Fasting GIP levels e . . . 61 5. GIP Serum Levels in Six Individuals Following Breakfast. . 64 6. GIP Serum Levels During Oral Glucose Tolerance Tests . . . 65 LIST OF FIGURES FIGURE PAGE 1. Antibody Response of Guinea Pig #76 to Immunization . . . 14 2. Column Profi le Early in Labelling Development . . . . . . 17 3. Column Profi le Following Gel F i l t rat ion Separation of Iodination Products on a 0.6 x 33 cm Sephadex G-15 Column 19 4. Column Profi le Following Iodination of 6 meg of GIP with 2 mCi of N a 1 2 5 I , Oxidized with 40 meg of Chloramine-T . . 21 5. Comparison of Variations in the pH of the Phosphate Buffer 23 6. A Comparison of the Effect on Standard Curve Sensit iv ity of Various Concentrations of Trasylol in the Diluent Buffer 25 7. A Comparison of Different Plasma Concentrations in the Diluent Buffer on Standard Curve Sensit ivity , 26 8. An Analysis of the Extent of ^ 5 j Self-absorption in Different Volumes of Diluent Buffer 28 9. Evaluation of Incubation Times to Determine Which Yielded Optimum Sensit ivity of the Standard Curve 29 10. A Comparison of Charcoal-Dextran, Methods for Separating Antibody/Antigen Complexes From Free Antigen 32 11. A Determination of the-Charcoal Concentration Required to Yield Optimum GIP/GIP Antibody Separation 33 12. Dilution Assay For Antibody Evaluation . , s , „ 36 13. Sensit ivity Assay Indicating Antibody Af f in i ty for GIP. . 38 14. Hook Effect Evaluated at Increasing Antibody Dilutions. . 40 15. Standard Curve Sensitivity Evaluation 46 16. Standard Curve Sensit ivity Evaluation 47 17. Standard Curve Sensit ivity Evaluation 48 FIGURE PAGE 18. Standard Curve S e n s i t i v i t y Evaluation . 49 19. Standard Curve S e n s i t i v i t y Evaluation . . . . 50 20. Evaluation of the C r o s s - S e n s i t i v i t y of S e c r e t i n , Glucagon, CCK-PZN and M o t i l i n to the GIP Antibody 51 21. S e n s i t i v i t y of the Standard Curve Based on Seven Determin-ations of Each Standard 52 22. A Replot of the Data of Figure Twenty One According to the Method of Nadeau and Zahnd Which Corrects f o r P r o t e i n I n t e r -ference 54 23. Linear P l o t of the E f f e c t of Plasma D i l u t i o n on Measured GIP Content of a Single Serum Sample D i l u t i o n 55 24. B/F of Serum D i l u t i o n F i t t e d to a GIP Standard Curve to Test f o r D i l u t i o n E f f e c t 56 25. A Comparison of the R e a c t i v i t y of GIP Standards and 10% CCK-PZN with GIP Antibody 58 26. A Q u a l i t y Control Chart Based on Ten Determinations of GIP Content of a 10% CCK-PZN Standard With 10% CCK-PZN S :,. - Standard Values of Subsequent Assays P l o t t e d . . 59 27. P r o b a b i l i t y Graph P l o t of the Frequency D i s t r i b u t i o n of GIP Normal Fasting Values Used to Determine the GIP Normal Range 62 28. Post P r a n d i a l Release of GIP 63 29. Serum GIP Levels During Oral Glucose Tolerance Tests, . . 66 ACKNOWLEDGEMENTS The problem i n c u r r e d w h i l e w r i t i n g acknowledgements i s thank-i n g c e r t a i n i n d i v i d u a l s w i t h i n t h e fo r m a l c o n t e x t o f a t h e s i s f o r a s s i s t a n c e w i t h o u t which t h i s t h e s i s would not have been c o m p l e t e d . My s i n c e r e a p p r e c i a t i o n goes t o Dr. J . Brown, who d e s p i t e f r e q u e n t i n c r e a s e s i n g a s t r i c a c i d i t y t h r o u g h o u t t h i s t h e s i s p r e p a r a t i o n p r o v i d e d e x c e l l e n t g u i d a n c e , s u p p o r t and f r i e n d s h i p . The a s s i s t -ance and p r e s e n c e o f M i s s J i l l D ryburgh was an immeasurable a s s e t . S p e c i a l t h a n k s must a l s o go t o my Mother f o r d e c i p h e r i n g my s p e l l i n g and t y p i n g t h e t h e s i s . I would a l s o l i k e t o acknowledge Kenten, who's appearance i n c r e a s e d t h e c h a l l e n g e . F i n a l l y , I would l i k e t o d e d i c a t e t h i s r e s e a r c h t o my husband, Ken; f o r h i s p a t i e n c e , s u p p o r t and p r o d d i n g . INTRODUCTION ] Investigation by Kosaka and Lim (1930a) established that a crude preparation of cholecystokinin (CCK-PZN) inhibited gastric secretion stimulated by a meat meal and histamine in dogs. They interpreted the results to indicate that the crude preparation was contaminated with an inhibitory material. They also obtained extracts of duodenal mucosa following stimulation with olive o i l 'which exhibited the same type of inhibit ion (1930b). Enterogastrone was the name given by them to the inhibitory material liberated by fat in the duodenum. This definition was extended by Gregory (1967) to "l iberation by fat digestion prod-ucts, hypertonic solution and acid of a hormone which inhibits gastric secretion and mot i l i ty . " Subsequently i t was found that crude CCK-PZN preparations inh ib i t -ed exogenous (Gillespie and Grossman, 1964) and endogenous (Brown and Magee, 1967) gastrin stimulated acid secretion. Pure CCK-PZN when i n -jected without gastrin, however, stimulated acid secretion. Brown and Pederson (1970) compared the response to 10% and 40% CCK-PZN prepara-tions in sympathetically and vagally denervated fundic pouches to at-tempt to resolve the confl ict ing responses. They found that 40% CCK-PZN stimulated acid and pepsin secretion to a greater extent than the . 10% preparation. During pentagastrin stimulation, 40% CCK-PZN result -ed in signif icantly lower inhibit ion of acid secretion. The effects on gall bladder and antral pouch moti l i ty were the same, There were two possible explanations for these results, Either an inhibitory material was being eliminated during the purification pro-cedures or a stimulatory substance was being concentrated. Since CCK-2 PZN and gastrin share many structural s imi lar i t ies i t appeared l ike l y that acid stimulation would be an inherent property of the CCK-PZN mol-ecule, the former hypothesis was pursued. Brown, Pederson, Jorpes, Mutt (1969) began purif ication of the possible inhibitor u t i l i z ing crude hog 10% CCK-PZN as the starting material. Assay of the starting material, designated EGI, in the guinea pig and cat indicated no secretin act iv i ty and less than 10 IDU CCK-PZN/mg, They obtained a signif icant inhibit ion of exogenous pentagastrin stim-ulated acid and pepsin secretion. Whereas 10% CCK-PZN stimulated an-tral motor act iv i ty , EGI was found to inhibit i t . Brown, Mutt and Pederson (1970) continued the purif ication of the polypeptide from EGI through to EGIII and named the material Gastric In-hibitory Polypeptide (GIP). Chemical evaluation was then init iated to determine the amino acid composition and sequence, The polypeptide was found to have a molecular weight of 5105. It consisted of forty three amino acids and the sequence was determined to be TYR-ALA-GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASP-LYS-ILE-ARG-GLN-GLN-ASP-PHE-VAL-ASN-TRP-LEU-LEU-ALA-GLN-GLN-LYS-GLY-LYS-SER-ASP-TRP-LYS-HIS-ASN-ILE-GLN (Brown, 1971; Brown and Dryburgh, 1971). GIP was chemically d ist inct from CCK-PZN by absence of proline, the presence of a high content of glutamic acid or glutamine and a pre-ponderance of lysine over arginine. Comparisons were also made with other gastrointestinal polypeptides, Structural s imi lar i t ies were found with both procine glucagon and secretin, GIP shares fifteen of the f i r s t 26 amino acids of glucagon and nine of secretin, Once GIP purification had proceeded to the EGI11 stage a series of 3 s t u d i e s were undertaken to determine i t s range of p h y s i o l o g i c a l a c t i v i -t i e s . Pederson (1971) compared the a c t i o n s of GIP on a c i d s t i m u l a t i o n by p e n t a g s t r i n and the whole g a s t r i n molecule (SHG-1). I n h i b i t i o n of a c i d and pepsin s e c r e t i o n and a n t r a l pouch motor a c t i v i t y were a l l g r e a t e r f o l l o w i n g SHG-1 s t i m u l a t i o n . Kosaka and Lim (1930a) found t h a t f a t i n the duodenum i n h i b i t e d histamine s t i m u l a t e d a c i d s e c r e t i o n . Studies with both CCK-PZN and s e c r e t i n (Johnson and Grossman, 1969) y i e l d e d no i n h i b i t i o n of histamine s t i m u l a t e d a c i d s e c r e t i o n and t h e r e f o r e n e i t h e r of them f u l f i l l e d the requirements f o r enterogastrone. I t t h e r e f o r e became important to look at GIP with r e s p e c t to histamine. Pederson and Brown (1972) determined the extent of i n h i b i t i o n of a c i d and pepsin s e c r e t i o n f o l l o w i n g h i s t a -mine s t i m u l a t i o n . They obtained r e s u l t s s i m i l a r i n degree to those obtained with f a t i n the duodenum (Johnson and Grossman, 1969, and A l l e y e t al_, 1934). The a c i d and pepsin s e c r e t i o n s t i m u l a t e d by i n s u l i n hypoglycemia was a l s o i n h i b i t e d by GIP. Barbezat and Grossman (1971a) compared the a b i l i t y of a s e r i e s of g a s t r o - i n t e s t i n a l p o l y p e p t i d e s to s t i m u l a t e s e c r e t i o n from i s o l a t e d loops of jejunum and ileum i n conscious dogs. GIP was a potent stim-u l a t o r of both j e j u n a l and i l e a l s e c r e t i o n . Although glucagon and pent-a g s t r i n were a l s o s t i m u l a t o r y they were much l e s s e f f e c t i v e than GIP, p a r t i c u l a r l y when comparing doses used on a molar b a s i s . CCK-PZN and s e c r e t i n were e s s e n t i a l l y without e f f e c t . In terms of the enterogastrone d e f i n i t i o n , GIP's a c t i o n s are much c l o s e r than any of the p r e v i o u s l y i n v e s t i g a t e d g a s t r o - i n t e r s t i n a l poly-p e p t i d e s . S e c r e t i n i n h i b i t e d a c i d s e c r e t i o n but s t i m u l a t e d pepsin 4 s e c r e t i o n (Naka j ima and Magee, 1970) , CCK-PZN e f f e c t s v a r y w i t h t h e p r e s e n c e o r absence o f exogenous g a s t r i n . More r e c e n t work by Grossman on the k i n e t i c s o f C C K - P Z N - g a s t r i n i n t e r a c t i o n i n d i c a t e s t h a t CCK-PZN i s a c o m p e t i t i v e i n h i b i t o r o f g a s t r i n / b o t h i n t e r a c t i n g w i t h a s i n g l e r e c e p t o r . When CCK-PZN was g i v e n a l o n e , t h e r e f o r e , i t a c t e d a t t h e r e -c e p t o r w i t h o u t c o m p e t i t i o n y i e l d i n g s t i m u l a t i o n , With exogenous g a s t r i n i n f u s i o n , h o w e v e r , t h e r e s u l t i n g i n h i b i t i o n o r s t i m u l a t i o n w i t h CCK-PZN depended on w h e t h e r CCK-PZN was as e f f e c t i v e as g a s t r i n i n s t i m u l a t i n g a c i d s e c r e t i o n . T h i s was a p p a r e n t l y s p e c i e s d e p e n d e n t . N e i t h e r CCK-PZN n o r s e c r e t i n i n h i b i t e d h i s t a m i n e s t i m u l a t e d s e c r e t i o n , G I P , however , i n h i b i t e d a c i d , p e p s i n and a n t r a l motor a c t i v i t y s t i m u l a t e d by g a s t r i n p e n t a p e p t i d e , g a s t r i n , i n s u l i n h y p o g l y c e m i a and h i s t a m i n e . I t appeared to c o u n t e r a c t a l l e f f e c t s o f g a s t r i n a c t i o n and mimicked the e f f e c t s o f f a t i n the duodenum. S i n c e GIP i n h i b i t e d t h e a c t i o n o f s t i m u l a t o r s f o r a c i d s e c r e t i o n o t h e r than g a s t r i n i t s a c t i o n was d i s t i n c t f rom C C K - P Z N . I t o h , L u c i e n and S c h a l l y (1972) have p r o p o s e d t h a t t h e i r " e n t e r o -g a s t r o n e " i n h i b i t s a c i d s e c r e t i o n a t the same s i t e as h i s t a m i n e , d i s -t i n c t f rom the g a s t r i n s i t e . T h i s e x p l a n a t i o n was u n t e n a b l e f o r t h e i r " e n t e r o g a s t r o n e " as w e l l as GIP because both i n h i b i t g a s t r i n s t i m u l a t e d a c i d s e c r e t i o n . Thus a p r o p o s a l must be made t o a c c o u n t f o r i n t e r -a c t i o n a t more than one r e c e p t o r . A l t h o u g h t h e " e n t e r o g a s t r o n e " o f I t o h y i e l d s s i m i l a r d e g r e e s o f i n h i b i t i o n o f g a s t r i n and h i s t a m i n e s t i m u l a t e d a c i d s e c r e t i o n i t i s i m p o s s i b l e , a t p r e s e n t , t o s ay i f the s u b s t a n c e s are i d e n t i c a l as no c h e m i c a l d a t a i s a v a i l a b l e on " e n t e r o g a s t r o n e " , The use o f 0 . 5 mg doses o f t h e I t o h m a t e r i a l t o o b t a i n i n h i b i t i o n sugges t s a f a i r l y low degree o f p u r i t y o r a v e r y low a c t i v i t y l e v e l . 5 It is not difficult to evaluate the physiological importance of such an inhibitor. If an enterogastrone was released by the presence of fat, acid and hypotonic or hypertonic solutions in the duodenum it would pro-tect the small intestine and stomach mucosa from excess H + secretion. Inhibition of acid would prevent further pH decreases and inhibition of antral motor activity would delay gastric emptying, The antral effect coupled with stimulation of intestinal secretion would improve absorp-tive functions in the small bowel. Once the inhibitory action of GIP had been evaluated, a means had to be developed for measuring GIP levels in blood and tissues, The availability of such an assay would enable the determination of. the physiological secretagogues for GIP release, This, would allow an eval-uation of the physiological role of GIP and also determine if it can be assigned hormonal status. Peptide hormones have characteristics invivo which differentiate them from the thyroidal and steriod hormones. They have a lower cir-culating basal level, which sporadically increase 1000% or greater over a short period of time. Their length of action is limited and thus the plasma one half life is usually less than thirty minutes. Chemically they cannot at present be separated from each other for rou-tine analysis. Attempts at bioassay to date have not yielded sufficient sensitivity to measure plasma levels. Even if a bioassay was developed, they are usually too tedious for routine clinical application. Similarity in the action and structure of the gastro-intestinal polypeptides was mentioned previously, There is also a considerable amount of interaction between peptides as indicated by CCK-PZN and 6 gastrin receptor act iv i ty . Thus with any assay u t i l i z e d , an evaluation of cross reactivity must be considered, Radioimmunoassays are well known for their sensit iv i ty and spec-i f i c i t y . Since the chemical reaction between antibody and antigen is extremely speci f ic , peptide antigens can be detected in the presence of a large number of other proteins without any prior extraction procedures, Specif icity can be evaluated for each antiserum ut i l i zed in the assay. Antibody and antigen concentrations can be reduced'proportionately to obtain sensi t iv i t ies in the low picogram range. The assay procedure is well suited for testing large numbers of samples at one time, thus making i t c l i n i c a l l y pract ical . The present research was undertaken to develop a radioimmunoassay, for GIP. The assay could then be ut i l i zed to evaluate GIP1s physiolog-ical role and possible pathological involvements. C l in ica l ly the only presently available GI peptide radioimmunoassay is for gastrin, A con-siderable amount of data on gastrin levels in pathological conditions has been published (Yalow and Berson, 1972) and the test is used diag-nostically in suspected cases of Zol1inger-Ellison Syndrome, 7 METHODS PRODUCTION OF ANTISERA I Immunization Schedule In i t ia l l y the procedure of Go, Ryan and Summerskill (1971) for CCK-PZN was used for immunization. Their schedule consisted of six subcu-taneous injections of 10% pure CCK-PZN at 10 day intervals (2.5 mg/ guinea pig) emulsified with Freunds Complete Adjuvant (FCA), Ten days following the last injection blood was taken by cardiac puncture. The antibody t i ters were boosted by subsequent immunizations with 50% puri -fied CCK-PZN (1 mg) in 1 ml of FCA on three occasions at ten- day inter -vals. GIP immunization schedules for six rabbits are found in Table 1 and ten guinea pigs in Table 2. The GIP side fraction (Brown, .Mutt and Pederson, 1970) used for i n i t i a l immunization was approximately 70% pure. Parker (1971) indicat-es that pure material is not required but one means of improving ant i -body af f in i ty is to increase the purity of the antigen with subsequent immunization. GIP III was thus substituted as immunization proceeded. Since the amount of antigen injected is an important variable in the production of antisera, this was also varied as the schedule pro-gressed. Exceeding the optimum amount can result in decreased antibody t i t e r due to complex formation invivo and may even induce tolerance, Insufficient antigen results in a deficient response (Odell, 1969), Throughout the assay development GIP was extremely scarce and as a re-sult a controlled study of antibody response versus antigen quantity could not be undertaken. The amount injected was decreased throughout 8 TABLE ONE IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO GIP IN RABBITS DAY INJECTION BLEEDING 0 0.5 mg GIP side A with FCA 22 0.5 mg GIP side A with FCA 12 days post marg, ear 56 0.5 mg GIP side A with FCA 13 days post card. punc 86 0.5 mg GIB side A with FCA 20 days post C P , 128 1.0 mg GIP side A with FCA 5 days post C, P , . 166 1.0 mg GIP II with FCA 10 days post C P . 183 1.0 mg GIP II with FCA 11 days post C.P, 226 0.5 mg GIP III with FCA 6 days post C P . 281 0.5 mg GIP III with FCA 11 days post C.P. 327 0.5 mg GIP III with FCA 12 days post marg. ear 420 0.5 mg GIP III with FCA 10 days post marg. ear 431 0.2 mg GIP III + BSA with FCA 10 days post marg, ear 482 0.2 mg GIP III + BSA with FCA 10 days post. marg. ear 9 TABLE TWO IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO GIP IN GUINEA PIGS DAY INJECTION BLEEDING 0 2.5 mg GIP side A with FCA 9 2.5 ng GIP side A with FCA 20 2.5 mg GIP side A with FCA 27 2.5 mg GIP side A with FCA 6 days post C P . 37 1.0 mg GIP II with FCA 7 days post C P . 44 1.0 mg GIP II with FCA 10 days post C P . 62 1.0 mg GIP II with FCA 16 days post C P . 82 1.0 mg GIP II with FCA 3 days post C P . 114 0.5 mg GIP III with FCA 6 days post C P . 136 0.5 mg GIP III with FCA 12 days post C P . 238 0.5 mg GIP III with FCA 11 days post C.P. 269 0.2 mg GIP III + BSA with FCA 11 days post C.P. 304 0.2 mg GIP III + BSA with FCA 17 -days post C P . 305 0.2 mg GIP 1111 + BSA with FCA 10 days post C P . 10 the schedule. At no time could an insuff icient response be attr ibut -able to decreased antigen, Thus at the end of the schedule, 0,2 mg of GIP III was found to be effective and was the optimum quantity chosen for GIP immunizations. The frequency of immunization yielding- the largest t i t e r increase varies for each antigen (Odell et a l j 1971), Unlike CCK-PZN (Go e^ t al_ f 1971) frequent boosters were not found to be effective with GIP, Deter-minations of the frequency of booster injections wi l l have to wait until a larger series of animals have been employed, At present animals are maintaining their level of antiserum three months post booster, Incorp-orating the above information, two new animals series were begun (Table 3), II Vehicle of Injection The GIP was dissolved in one volume of saline which was emulsified with an equal volume of Freuhdsreoraplete/'Adjuvant.(BAC). The FAC. was employed to reduce the rate of release of antigen thus sustaining the stimulus to antibody production. To obtain an:' even slower rate of re-lease the saline to FCA ratio was changed to 2/3 (Hurn and Landon, 1971). III Injection Route and Volume Originally a single subcutaneous injection of 1,0 ml was given to the rabbits and 0.5 ml to the guinea pigs.- The volume was later reduc-ed to two 200 jul subcutaneous injections at different s i tes , This re-duced the local reaction sores. Although sores are the result of a local inflammation and therefore, indicative of a good response, the sores were severe and threatened the preservation of the animals, With a 200 TABLE THREE 11 IMMUNIZATION SCHEDULE FOR'THE .'INDUCTION OF ANTIBODIES TO A NEW SERIES OF ANIMALS (7 GUINEA PIGS) DAY INJECTION BLEEDING 0 0 . 5 mg GIP I I I i n . FCA 29 0 . 2 mg GIP I I I + BSA i n FCA 70 0 . 2 mg GIP I I I + BSA i n FCA .13 days post IMMUNIZATION SCHEDULE FOR THE /INDUCTION OF ANTIBODIES TO.GIP IN GUINEA PIGS " . DAY INJECTION BLEEDING 0 ; 1 . 0 mg GIP II w i t h FCA 135. '0 .5 mg GIP' I I T w i th FCA 258 0 . 2 mg GIP I I I + BSA w i th FCA 11 days post C . P . 290 0 .2 mg GIP I I I + BSA w i th FCA 10 days post C . P . 341 0 . 2 mg GIP I I I + BSA w i th FCA 10 days post C . P . 12 jul volume sores were s t i l l observable but did not require treatment, The order of increasing effectiveness of injection sites i s : int ra -venous, subcutaneous, intraperitoneal, intramuscular, intradermal, intra art icular (knee joint) and lymphatic nodes (Hum and Landon, 1971) Due to the relative ease of subcutaneous injections this route was retained, Ut i l i z ing two injection sites in place of a single one, however, better distribution to the lymph nodes of both back and abdomen was obtained, Another factor favoring the continued use of back and abdominal injection-sites is that the sensitized ce l ls formed during the i n i t i a l immunization tend to localize in lymph nodes near the si te of injection, Thus subsequent injections are most effective when given to the same area (Parker, 1971). IV Conjugation Following 11 injections even the best animals were producing ant i -serum of only low t i t e r . Although Yalow and Berson (1970) indicate that a 5,000 molecular weight protein should be antigenic on i ts own, conjuga-tion to bovine serum albumin (BSA) was tested. Conjugation was obtained by activation with CDI (I-ethyl-3-[3-dimethyl-aminipropyl]-carbodimide), which form CO-NH bonds. CDI is water soluble and has an optimum pH of 5.5 (Parker, 1971). The method used for conjugation was that of Cataland (personal communication). Forty mg of bovine serum albumin, 1,5 mg of GIP IV and 400 mg of CDI were dissolved separately i n , respectively 1,0 ml, 500 jjl and 500 jjl of deionized water, The reagents were then added to the GIP solution and mixed thoroughly, Following incubation for one hour at room temperature, the mixture was dialysed against deionized water 13 at 4°C. At the end of 24 hours, 10 |il of the dialysate was removed for a high voltage run at pH 2 ,1 , and the remainder was lyophil ized, Although the optimum pH for CDI activation is 5,5, the procedure indicated ut i l i zes a pH of 7.0. Sufficient time was not available for evaluating the best pH for GIP conjugation. One factor in favor of pH 7.0 is that i t would reduce the degree of protein dimerization, which can be a problem with carboxyl group activation (Parker, 1971), The i n i t i a l response to conjugation was str iking as evaluated by t i t e r increase. Subsequent booster shots yielded one further increase followed by a plateau after the second injection (Figure 1), Since BSA wi l l also be inducing antibody formation, further boosters consisted of unconjugated GIP. One important finding from the motilin radioimmunoassay was that conjugate was most effective when preceded by unconjugated antigen. Thus new GIP immunization schedules were begun with two GIP III injec-tions prior to the BSA conjugation (Table 3). V Bleeding and Treatment of Antisera In i t ia l l y a l l bleeding was by cardiac puncture but due to fa ta l i t ies the rabbits were bled by marginal ear vein. Holding the guinea pigs securely and bleeding them only i f the heart was punctured on the f i r s t attempt was found to reduce mortality. Serum was obtained by centrifug-ing the samples for 15 minutes at 4,000 rpm, I n i t i a l l y the serum was aliquoted into approximately 100 ^ il samples which were frozen in dry ice and methyl cellosolve and stored at -20°C, No preservative agents were required. Serum has been used without loss o £ o_ t—* o Q. t—I 200 Time in Days 300 FIGURE ONE: Antibody response of guinea pig #76 to immunization. 15 of a f f in i t y for periods as long as six months. Once the 100 JJI aliquots had been thawed they were realiquoted i n -to 10 ^ il quantities, and refrozen;, A 10 /ul aliquot was diluted up to 1.0 ml for preparing an individual assay's antibody. The 1 ml di lut ion reduced the amount of gamma globulin adsorbed to the tube yielding greater consistancy of f inal di lut ions. Lyophilization of serum was considered but the l i terature reports confl ict ing results as to the s tab i l i t y (Hum and Landon, 1971). Since there was no problem in s tab i l i t y with freezing, i t was considered the method of choice. LABELLING OF GIP The original labell ing procedure involved the addition of the f o l -lowing reagents in rapid succession to 5 jjg of GIP IV in 100 JJ1 of 014M PO^  buffer pH 7.5 (Deftos, personal communication to D.H. Copp): REAGENT VOLUME AMOUNT 1. N a l 1 2 5 5 J J I 1 mCi 2. Chloramine-T 20 J J I in D.I. water 176 jjg 3. Sodium metabisulphite 20 /ul in D.I. water 252 jug Steps 1-3 are performed with bubbling to insure proper mixing. 4. Quso suspended in 10 mg 1 ml 0.4M P0 4 Mix on vortex mixer and spin down. 5. 40% acetone/acetic acid 1.0 ml 16 Mix on vor tex 6 , Dowex Mix and s p i n down. The supernatant was decanted and d i v i d e d i n t o a l i q u o t s f o r f u r t h e r p u r i f i c a t i o n on a column, A l i q u o t s were s to red a t -20°C u n t i l used a t which t ime they were run on a 0 .6 x 22 cm G25 Sephadex column, The D e f t o s - method y i e l d e d adequate l a b e l l e d GIP f o r ant ibody e v a l u a t i o n a s s a y s , M o d i f i c a t i o n had to be i n t r o d u c e d , however; to y i e l d a ^^l-GIP product s u i t a b l e f o r s tandard c u r v e s , A bas i c m o d i f i c a t i o n was the p u r i f i c a t i o n of the l a b e l l e d product on a Sephadex G25 column (0 .6 . x 22 cm) immediately f o l l o w i n g i o d i n a t i o n . Although, pr imary i n t e r n a l r a d i a t i o n damage (decay of I) cannot be a v o i d e d , pr imary e x t e r n a l (decaying ]25j . i n t e r a c t i n g w i th other l a b e l l e d p r o t e i n molecu les ) and secondary (secondary ions produced t.n>;theG]'iqui'<ki medium by. gamma rays ) can be reduced by e l i m i n a t i n g as q u i c k l y as p o s s i -b le a l l unreacted 1 2 5 I and l a b e l l e d pept ide f ragments . Thus the Quso and Dowex were s u b s t i t u t e d by immediate gel f i l t r a t i o n on the.Sephadex column. The f r a c t i o n s were counted on a gamma counter (Model 4227, Nuc lear Ch icago ) , Counts/minute were p l o t t e d a g a i n s t t u b e . f r a c t i o n s to y i e l d a column p r o f i l e . A t y p i c a l p r o f i l e a t t h i s stage i s shown, in F igure 2 , The f i r s t peak was the l a b e l l e d GIP. molecules and fragments w h i l e the second peak i s unreacted I. The a c t i v e peaks were a l i q u o t e d i n 100 jul q u a n t i t i e s and f r o z e n . Both procedures reduce.secondary r a d i a t i o n . damage, A method.had to be d e v e l o p e d . f o r t e s t i n g the column f r a c t i o n s to 6 0 0 N to 2 4 0 0 CD Z3 C / \ *' X x / I x-x X X O 2 0 0 \ c o o 4 - r 8 12 16 20 24 Column Fractions 400/JI.Aliquots FIGURE TWO: Column profile early in labelling development. 18 determine which contained the immunoreactive ^ I - G I P , The laboratory f a c i l i t i e s precluded the use of chromatoelectrophoresis (Yelow and Ber-son, 1960) so a simple incubation in excess antibody was chosen and re-ferred to as a damage assay. Essentially the assay evaluated the frac-tions on their ab i l i t y to react with antibody compared with their non specif ic interaction with the di lut ing buffer, To improve peak separation a 33 cm Sephadex G-15 column was sub-stituted for the 22 cm G-25. Figure 3 is the resulting column prof i le indicating a marked increase in peak separation. At this time, the eluting agent was the same 0,04 PO^  buffer used 125 for the assay. I-GIP storage l i f e was approximately ten days. To increase this time 0.2M acetic acid was substituted for the phosphate buffer (Hunter, 1971). Stored in the acetic acid the shelf l i f e of labelled GIP was increased to three weeks, Another area of concern was with the use of chloramine-T, a power-ful oxidizing agent used to convert iodide to iodine during label l ing, Preliminary evaluation with polyacrylamide gel electrophoresis ind i -cated GIP molecules undergo fragmentation with exposure to chloramine-T. Subsequent evaluation indicated that the chloramine-T acted on the trytophan in GIP (Brown, personal communication) and thus the reaction was specific to some extent to peptides containing this amino acid. This sensit iv i ty of GIP to chloramine-T eliminated the possibi l i ty of improving iodination by reducing the iodination volume (Hunter, 1971), since this increased considerably the chloramine-T concentration during iodination. The alternative available for improving "^I-GIP specific act iv i ty was to alter the GIP/chloramine-T/Na 1 2 5I rat ios, Labellings Column Fractions : 400p\. Aliquots I ' i FIGURE THREE: Column profi le following gel f i l t ra t ion separation of iodination products on a 0.6 x 33 cm Sephadex G-15 column. 20 were eva luated w i t h 5/176/1, 5/50/1, 5/50/2, 5/35/2. 5/25/2, 5/40/2 and 6/40/2 (fig/jig/mCi) r a t i o s . The f i n a l r a t i o o f 6/40/2 was found to r e -s u l t i n the h ighes t precentage y i e l d of immunoreactive GIP p lus the low-e s t non s p e c i f i c b i n d i n g of the a c t i v e peaks (F igure 4 ) . The immunore-a c t i v i t y and non s p e c i f i c b i n d i n g were determined by a damage assay and the percentage y i e l d eva luated from the counts per minute of 10 y l a l i q u o t s o f column f r a c t i o n s . Another i n d i c a t i o n of improved i o d i n a t i o n was the i n c r e a s e i n usab le f r a c t i o n s . I n i t i a l l y two f r a c t i o n s near the peak were immunoreact ive. With the new r a t i o a l l f r a c t i o n s from the peak and on the descending l imb cou ld be u t i l i z e d i n a s s a y s . The 6/40/2 r a t i o was t h e r e f o r e optimum f o r GIP l a b e l l i n g . The l a b e l l i n g procedure f o r GIP IV was then m o d i f i e d t o : GIP IV t h a t had been p r e v i o u s l y d i v i d e d i n t o 6 /jg a l i q u o t s and l y o p h i l i z e d , was used f o r l a b e l l i n g . A s i l i c o n i z e d tube (12 x 75 mm) comta in ing 6 pq of GIP IV was r e c o n s t i t u t e d by the a d d i t i o n of 100 j j l o f 0 .4 P 0 4 b u f f e r a t pH 7 . 5 . The whole l a b e l l i n g procedure was c a r r i e d out i n one tube to prevent a d s o r p t i o n l o s s e s . Added i n the order l i s t e d were: 1) 2 mCi of 1 2 5 I 20 jul 2) 40 jjg of ch loramine -T - - - - - - - 10 J J I 15 second t ime d e l a y . 3) 252 yg o f sodium m e t a b i s u l p h i t e - - 20 jul Steps were performed w i t h bubb l ing to i n s u r e proper mix ing and reagents added as q u i c k l y as p o s s i b l e except where i n d i c a t e d . A f t e r complet ion of the l a b e l l i n g the 150 J J ! r e a c t i o n volume was added immediately to a Sephadex column (0 .6 x 33 cm). The column was Column Fractions - 10 /j| Aliquots FIGURE FOUR: Column prof i le following iodination of 6 meg of GIP with 2 mCi of Na f oxidized with 40 meg of chloramine-T. 22 e l u t e d w i t h 0.2M a c e t i c a c i d b u f f e r t o w h i c h had been added 1% human plasma ( to r e d u c e p e p t i d e a d s o r p t i o n t o the co lumn) and 2% T r a s y l o l (25 ,000 KIU/5 m l , a p r o t e a s e i n h i b i t o r ) . A p p r o x i m a t e l y 40 t u b e s o f 8 d r o p s / t u b e ( a p p r o x i m a t e l y 300 u l ) were c o l l e c t e d and 10 u l a l i q u o t s were c o u n t e d . Once t h e f r a c t i o n s were c o u n t e d t h e a c t i v e tubes were d i v i d e d i n t o 50 u l a l i q u o t s , f r o z e n i n d r y i c e and methyl c e l l o s o l v e , and s t o r e d a t - 2 0 ° C . The 10 u l a l i q u o t s were used t o s e t up a damage a s s ay t o e v a l u a t e t h e l a b e l l i n g . STANDARDS Two mg o f GIP IV were weighed a c c u r a t e l y on a m i c r o b a l a n c e (Cahn) and d i s s o l v e d i n 3 3 . 3 ml o f d e i o n i z e d w a t e r . Two hundred q u a n t i t i e s (6 ug) were a l i q u o t e d i n t o v i a l s ; w h i c h were l y o p h i l i z e d , s e a l e d and s t o r -ed a t - 2 0 ° C u n t i l r e q u i r e d . CONDITIONS OF ASSAY I . D i l u e n t B u f f e r The s t a n d a r d d i l u e n t b u f f e r used i n the a s s a y i s 0.04M P0^ pH 6 .5 w i t h 5% T r a s y l o l ( S . B . A . P h a r m a c e u t i c a l L t d . ) and 10% o u t d a t e d human p l a s m a . A l l b u f f e r v a r i a b l e s were t e s t e d s e p a r a t e l y t o d e t e r m i n e o p t i -mum a s s a y c o n d i t i o n s . Phosphate b u f f e r s a t p H ' s 6 . 5 , 7 .5 and 8 . 5 were t e s t e d and pH 6 .5 y i e l d e d t h e b e s t s t a n d a r d c u r v e ( F i g u r e 5 ) . T r a s y l o l p r e v e n t s p r o t e a s e a c t i v i t y thus p r o t e c t i n g p e p t i d e s . O r i g -i n a l l y 1% (5 ,000 K i l l / m l ) was added t o t h e d i l u e n t b u f f e r . I t was sub-s e q u e n t l y found t h a t 5% i n c r e a s e d t h e s e n s i t i v i t y o f t h e s t a n d a r d c u r v e . x x II 8.5 J 3 F 0.80 ^  G I P JSL S t a n d a r d s in ng . FIGURE FIVE: Comparison of variations in the pH of the phosphate buffer. 24 Figure 5 shows standard curves with 0, 2,5, 5 and 10% Trasylol in the diluent buffer. Sensit ivity was increased when 5% Trasylol was used and depressed with 10%. The 10% depression must be due to.an action of Tra-sylol on the GIP molecule. The results of this study and others ind i -cated that 5% Trasylol produced the maximum protection without a sup-presive action. This was supported also by an assay comparing 0, 2,5 and. 5% Trasylol in diluent buffer used for standard curves evaluating known serum samples. Again 5% yielded the best results. Plasma addition also had to be evaluated. Standard curves were set up to compare the effect of varying concentrations of plasma in the diluent buffer. The 2% and 10% curves are shown in Figure 7 indicating the increased sensit iv i ty of the curve (evaluated by the slope) with 10% human plasma. The plasma used in the assay is outdated blood bank plasma, Orig-ina l ly i t was added without treatment as polypeptides and protease ac-t i v i t y should be short l ived.' Routine screening of the new plasma in the assay indicated readable GIP levels , however, so an overnight char-coal extraction at 4°C (1.0 mg charcoal/300 ml plasma), is now routine procedure. II Reaction Volume The standard reaction volume for the assay was 0.5 ml. Larger vo l -umes were tested but no gain in sensit iv ity was found. This volume was used as i t conserved antibody and provided for best counting characteris-t i c s . 125j decays by very weak gamma radiation. This low energy level can be absorbed by the solution (self-absorption) and thus the larger the counting volume the lower the number of counts obtained. JSince both the FIGURE SIX : A comparison of the effect on standard curve sensit iv i ty of various concentra-tions of Trasylol in the diluent buffer, _B F Standard GIP in ng cr-FIGURE SEVEN: A comparison of different plasma concentrations in the diluent buffer on standard curve sensit iv i ty . 27 precipitate and supernatant are counted, a difference in volume could prejudice the count rate in favor of the smaller volume, An analysis of volume effects on count rate (Figure 8) indicated that within the range of 0,1 to 1,5 ml the counts were within one S.D, Thus to reduce error due to volume differences the precipitate and supernatant should be within the 0.1 to 1.5 ml range. III Assay Tubes The reaction volume was incubated in glass culture tubes (Kimble, 10 x 75 mm). In i t i a l l y the tubes were used without s i l i con iz ing . When 100 jul of antigen was added alone to nonsiliconized tubes, and le f t to stand over the incubation period only 1/3 of the radiation could be separated out by dioxane. This indicated a very high degree of tube absorption. It was eliminated to a great extent by s i l iconizat ion, It also allowed for greater ease in decanting the supernatant following centrifugation, thus reducing the amount of supernatant l e f t in the pre-c ip i tate . IV Incubation Time A comparison of different incubation times was undertaken, Equi-librium assays for 24, 48, and 72 hours were compared to disequilibrium assays with a 24 and 48 preincubation without antigen followed by 24, 48 and 72 hour post antigen addition incubation, Figure 9 i l lustrates the affect of incubation time on the standard curve, The 24 hour pre and 48 hour post disequilibrium and 48 hour equilibrium yielded similar results, Prior to the use of this antibody at the present 1/40,000 d i lu t ion , the disequilibrium assay was far superior, Since the 48 hour FIGURE EIGHT: An analysis of the extent of '"I self-absorption in different volumes of diluent buffer. 2 9 ! x — x 24 pre cold' 48 post cold 0 = 0.077 ! a—A 24 pre hot 48 post cold D=0.I66 • — • 24 Equilibrium D = 0.078 o—o 48 " DO.098 F 0.8 0 -, 0.00+ , . 1 10 100 GIP Standard in pg FIGURE NINE:. Evaluation of incubation times to determine which yielded optimum sensit iv ity of the standard curve. 30 e l i m i n a t e s o n e d a y i t was p r e f e r r e d o v e r t h e d i s e q u i l i b r i u m a s s a y , a n d was u s e d i n s u b s e q u e n t a s s a y s . V) A s s a y P r o c e d u r e T h e o p t i m u m c o n d i t i o n s o f t h e a s s a y f o r GIP w e r e f o u n d t o b e : 1) T h e t o t a l i n c u b a t i o n v o l u m e was 0,5 m l , A n t i b o d y , ^ 2 5 I - G I P , s t a n d a r d s a n d unknown w e r e d i l u t e d w i t h d i l u e n t b u f f e r (0,04M PO4 pH 6,5 w i t h 1 0 % human p l a s m a a n d 5% T r a s y l o l ) t o y i e l d t h e d e s i r e d c o n c e n t r a - 5 t i o n i n a 100 j u l v o l u m e . T h e v a r i o u s c o m p o n e n t s o f t h e a s s a y w e r e a d d -e d w i t h 100 j u l s a m p l e r p i p e t s ( O x f o r d L a b o r a t o r i e s ) a n d t h e v o l u m e was a d j u s t e d t o 0.5 ml by a d d i t i o n o f d i l u e n t b u f f e r v i a an O x f o r d P i p e t o r ( M o d e l NO, 1 3 - 6 8 7 - 9 0 ) . T h e t u b e s ( s i l i c o n i z e d , g l a s s c u l t u r e t u b e s , K i m b l e 10;x 75 mm) w e r e t h e n m i x e d b r i e f l y on a V o r t e x - G e n i e ( S c i e n t i f -i c I n d u s t r i e s I n c . , M o del K 5 5 0 - 6 ) a n d i n c u b a t e d a t 4 ° C . 2) A l l s t a n d a r d c u r v e s w e r e s e t up as e i t h e r d i s e q u i l i b r i u m a s s a y s w i t h a 24 h o u r p r e i n c u b a t i o n o f t h e a n t i b o d y a n d s t a n d a r d o r unknown a n d a 48 h o u r p o s t i n c u b a t i o n f o l l o w i n g a n t i g e n a d d i t i o n , o r a 48 h o u r e q u i l i b r i u m a s s a y . 3) C o n t r o l m i x t u r e s c o n t a i n i n g no a n t i b o d y w e r e s e t up f o r a l l s a m p l e s t o p e r m i t c a l c u l a t i o n o f i n c u b a t i o n damage, A l l s a m p l e s w e r e a s s a y e d i n t r i p l i c a t e . 31 SEPARATION PROCEDURES Three basic separation procedures have been employed to separate the GJ.P-antibody complex from the unreacted GIP: dioxane precipitation, double antibody and peptide adsorption, In i t ia l l y dioxane (a diethylene dioxide which denatures and prec-ipitates the antibody-antigen complex) was ut i l i zed but i t became i n -creasingly apparent that i t was not adequate for separation. As the amount of plasma in the assay was increased, the amount of nonspecific precipitation increased to a level incompatible with assay sensi t iv i ty , This was also reported by Thomas ert al_ (1969), A series of assays were then set up to find a better means of sepa ration. Gastrin (a negatively charged polypeptide) had been separated with amberlite, an anion exchange resin (Yalow and Berson, 1970), At a pH of 6.5 GIP is positively charged and so cationic exchange resins were evaluated f i r s t . As was feared the antibody was also positive at pH 6.5 and thus CM11 and SE Sephadex both provided inadequate separa-t ion . Separation on the basis of size with Sephadex G-50, quso and talc were also inef f ic ient . An assay was performed to compare various quantities of charcoal and dextran coated charcoal with dioxane separation (Palnier i , Yalow and Berson, 1971). Both separation procedures were superior to diox-ane (Figure 10), Charcoal-dextran was superior to charcoal alone due to a lower damage and greater consistancy of results with increasing plasma concentrations. As a result of a series of experiments (Figure 11), 2.5 mg of charcoal with 0.5 mg of dextran per tube was found to be optimum. _B_ F 1.40 A Charcoal - Dextran (5mg) o o Charcoal (5mg) x x Dioxane ( I ml ) Damage Assays of each 8 12 % Plasma in Diluent Buffer 20 FIGURE TEN: A comparison of charcoal-dextran, methods for separating antibody/ antigen complexes from free antigen. 1.40 1.20 I .00 0.80 H 0.60 0.40 • A A 5 rngm Charcoal / Tube o o 2.5 " " " Damage 0.20 0.00 _~_—-----8 20 24 28 34 36 40 0 4 12 16 % Plasma in Diluent Buffer FIGURE ELEVEN: A determination of the charcoal concentration required to y ield optimum GIP/GIP antibody separation. u> 34 The c h a r c o a l - d e x t r a n was added to the assay in 200 jul of d i l u e n t b u f f e r (pH 6 . 5 0.04M P 0 4 b u f f e r w i t h 2% human plasma and 1% T r a s y l o l ) Th is was h igher than the maximum 20% of assay volume suggested by P a i n i e r i et aj_ (1971) , but i t was found to be the minimum volume r e q u i r -ed f o r adequate s e p a r a t i o n of p r e c i p i t a t e and supernatant , The sug -g e s t i o n of v a r i o u s workers (Hunter and G a n g u l i , 1971) to add 1 ml of d i l u e n t b u f f e r j u s t p r i o r to c e n t r i f u g i n g , reduced r a t h e r than improved the r e s u l t s . One f u r t h e r m o d i f i c a t i o n o f the s e p a r a t i o n procedure was i n t r o -duced. The c h a r c o a l - d e x t r a n was made up j u s t p r i o r to use , Arnaud (1971) prepares the mixture the n ight before and leaves i t mix ing o v e r -n i g h t . Th is procedure was found to y i e l d improved assay s e n s i t i v i t y , A comparison was a l s o made wi th the double ant ibody method, U t i l -i z i n g the method and m a t e r i a l s of Cata land (personal communication) f o r double ant ibody s e p a r a t i o n , c h a r c o a l - d e x t r a n s e p a r a t i o n was found to y i e l d s u p e r i o r s e p a r a t i o n . The s e p a r a t i o n procedure adopted was: the day p r i o r to s e p a r a t i o n of the assay the c h a r c o a l - d e x t r a n was prepared . The b u f f e r (0.04M P0^ b u f f e r pH 6 . 5 w i t h 2% human plasma and 1% T r a s y l o l ) was coo led p r i o r to the a d d i t i o n of the dext ran (T 7 0 , Pharmacia Fine C h e m i c a l s ) , Once a dextran s o l u t i o n had been o b t a i n e d , the charcoa l (carbon d e c o l o r i z i n g n e u t r a l C - 1 7 0 , F i s h e r ) was added. The amounts added y ie lded , a f i n a l c o n c e n t r a t i o n o f 2 . 5 mg o f charcoa l and 0 . 5 mg of dext ran f o r each 200 /jl o f b u f f e r . The mixture was l e f t to s t i r on a mag mixer overn ight at 4°C. In the morning a h a l f hour t i m i n g was s t a r t e d w i t h the f i r s t 200 j j l 35 addition of the charcoal-dextran to a tube, After completing the addi-tion of the mixture to the assay, the tubes were vortexed and le f t to stand at 4°C until the half hour was completed, The tubes were then centrifuged at 2800 rpm for 15 minutes and the supernatant was decanted, The tubes were corked and placed into plastic carrier tubes for counting, TYPES OF ASSAYS There were basically four types of assays set up which tested, antigen labell ing (damage assay), antibody bleeding (dilution assay), antibody-antigen reaction (sensit ivity assay), and GIP levels (standard curves). I Damage Assay This assay consisted of two types of tubes, One contained only diluent buffer and antigen. This was a measure of the nonspecific bind-ing of the antigen (adsorption and nonspecific protein interactions), The second tube contained a certain constant di lution of antibody and measured the maximum antibody-antigen binding. This assay was set up after each new label l ing to evaluate the quality of the new labelled material. II Dilution Assay Damage tubes were set up as correction factors, Instead of varying the antigen, however, different antibody dilutions were included to find the di lution at which 30 to 50% of the antigen was bound. These assays were usually set up .for a quick evaluation of a new bleeding (Figure 12), F I .80 A • • 1 3 / 6 / 7 2 Bleeding o o 1 2 / 5 / 7 2 i I /1000 Antiserum final Dilution 1 1/10000 FIGURE TWELVE: Dilution assay for antibody evaluation. Vu 37 III Sensit ivity Assay In this assay both antibody and antigen concentration were var ia-ble. This tested the af f in i ty of the antibody for antigen and also yielded exact information on optimum antibody and antigen concentration. Figure 13 indicated that the antibody can distinguish between the addi-tion of antigen yielding 40,000, 20,000 and 10,000 cpm. This type of assay was usually used during the development stages of the assay. Once standard curves were available evaluations comparing curve sensit iv i ty were more profitable. IV Standard Curves Sensit ivity (considered to be either the slope of the standard curve or the lowest standard which can signif icantly be distinguished from zero competition) was the key to the standard curve. Once a l l the assay conditions had been standardized this depended primarily on the energy of the antibody/antigen reaction. Once a reasonable t i t e r (final dilution usable in the assay) was reached, increasing the sensit iv i ty required an increase in the af f in i ty of the antibody for the antigen. Changes in antigen labell ing as well as antibody avidity affected this reaction. Once the sensit iv i ty of the assay was established in the low pico-gram range one inconsistent problem had to be evaluated. In many of the standard curves the low picogram standards yielded higher B/F's than the zero competition (hook effect) . Immunologically the hook effect could be explained on the basis of excess antibody. If this were the case, in the upper part of the curve where antigen quantities were low-est; less complex antibody/antigen formations would occur. Charcoal-_B_ F 1.40 1.20 i.oo H 0.80 H 0.60 0.40 0.20 H 0.00 Antigen (Cpm' k—A 4 3 0 0 0 3 ° 2 9 0 0 0 3 — ° 1 2 5 0 0 1/100 1/1000 Guinea Pig final Dilution l/IOOOO FIGURE THIRTEEN: S en s i t i v i t y assay indicat ing antibody a f f i n i t y for GIP 39 dextran separation would be less eff ic ient with the smaller complex thus prejudicing the free component and yielding a decreased B/F, To test this hypothesis, standard curves were set up with f inal dilutions of 1/20,000. 1/30,000 and 1/40,000 (Figure 14), The hook effect disappear-ed with the 1/40,000 dilution thus supporting the theory, The elimination of the hook effect at a 1/40,000 f inal di lution results in a zero competition B/F in the range of 0.7 to 0,5. This is in opposition to the generally held concept that assays should aim for 50% binding or a B/F of 1.0. For GIP a B/F of 0.5 yields a superior standard curve and was used routinely, Another standard practice is the addition of 10,000 cpm of labelled antigen to assays. This practice does not take into consideration d i f -ferences in specific act iv i ty of the labelled material. 10,000 cpm could mean the addition of 100 pg or 10 pg depending on the extent of iodine substitution of the peptide. Yalow and Benson (1971a) indicate that one means of improving the sensit iv i ty is to decrease the amount of antigen added. This decrease is l imited, however, by the, increasing counting errors introduced i f adequate total counts are not collected. Also i f the antibody is not of suff icient sensit iv i ty to distinguish between 100 pg and TO pg of antigen, a reduction of labelled antigen to the 10 pg range wi l l not y ie ld an increase in sensit iv i ty . With GIP the counts added to the assay depends on the efficiency of the label -l ing and the shelf l i f e of the material. With a new labell ing a reduc-tion to 4,000 cpm (approximately 10 pg) w i l l improve sensi t iv i ty , As the label' ages, the labelled fragments increase and 8,000 cpm was the lowest practical leve l . This occurs at about two and one half weeks, SOURCE OF HORMONE PREPARATIONS 41 S y n t h e t i c Human G a s t r i n (SHG-15-Leu): Max-Planck I n s t i t u t e f u r E i w e i s s und Lederforschung (Munich). CCK-PZN (250 I.D.U./mg and 1500 I.D.U./mg): G.I.H. Research L a b o r a t o r i e s (Stockholm, Sweden). S y n t h e t i c S e c r e t i n : Max-Planck I n s t i t u t e f t l r E iweiss und Lederforschung (Munich). Natural Porcine S e c r e t i n : G.I.H. Research L a b o r a t o r i e s (Stockholm, Sweden). S y n t h e t i c Glucagon: Max-Planck I n s t i t u t e f t i r Eiweiss und Lederforschung (Munich). ANALYSIS OF DATA There are numerous methods a v a i l a b l e f o r c a l c u l a t i n g and expres-s i n g assay r e s u l t s . Methods t e s t e d f o r GIP i n c l u d e : CPM bound, percent bound (using zero competition as 100%), Y (where Y equals the bound of the standard/bound of zero competition) and B/F. Each r e s u l t was p l o t t e d a g a i n s t the log of the standard c o n c e n t r a t i o n . Only the B/F c a l c u l a t i o n i n v o l v e s counts of both p r e c i p i t a t e and supernatant. T h i s double count c o r r e c t s and/or c o n t r o l s f o r unequal antigen a d d i t i o n , incomplete s e p a r a t i o n of p r e c i p i t a t e and supernatant and counting e r r o r s . The B/F c a l c u l a t i o n a l s o i n v o l v e s a c o r r e c t i o n f a c t o r D (damage) f o r non s p e c i f i c b i n d i n g to g l a s s or plasma p r o t e i n s . The formula used f o r B/F c a l c u l a t i o n s was: B/F = B - (B + F) x 0.D where D = B d i n damage tubes (Deftos, personal communication to D.H. Copp). A l l serum samples were c o r r e c t e d with t h e i r separate damage tubes c o n t a i n i n g the i n d i v i d u a l serum t e s t e d . 42 To determine the sensitivity of the standard curve; the Student t test and Fisher's modification of the Student t test were used to deter-mine the significance of the difference between the means of two small non correlated samples, The sensitivities indicated during the eval-uation of standard curve improvement were determined as 10% of the low-est readable standard concentration. This was used in place of the Fisher t test (Smith, 1964) due to lack of adequate statistics on e a r l i -er curves. This approach can be j u s t i f i e d , in that case, as the values were used as qualitative comparison of standard curve improvement and not for evaluating test samples. Inter and intra assay precision determinations were based on the coefficient of variation which, expressed as a percent, is equal to the standard deviation divided by the mean, times 100. A value of 10% or less is considered to be necessary for radioimmunoassays (Loraine and Bell, 1971). The normal ranges and quality control charts were set up according to the procedures of Hoffman (1971). The limits of the quality control chart were based on +2 S.D. of 20 repeat values of the 10% CCK-PZN standards. Normal ranges were based on the averages-of-normals method, A cummulative distribution was determined followed by a calculation of the cummulative percent. The data was then plotted on normal probabil-its graph paper from which the normal range could be determined, Utilizing the Nadeau and Zahnd (1971) correction for damage and non specific protein interaction, Ba/F ratios were obtained: = Bo - Be F Be-Bb Be-Be Where: Bo = Bound cpm for excess antibody tubes, Be = Bound cpm for unknown or standard, and Be = Bound cpm for damage tubes. 43 The specific act iv i ty of the labelled GIP was determined, The evaluation was based on the percent of act iv i ty corresponding to immuno-reactive 1 2 5 I -G IP fractions in a column profi le (Figure 4), This is a deviation of the method reported (Hunter, 1971) which estimates the number of 1 2 5I molecules substituted/polypeptide molecule,, Both methods are rough approximations. Specific Activity of 1 2 5I used: 14 mCi/mcg Therefore in 1 mCi there are 72 ng 143 ng of '"I were reacted with 6 meg of GIP On a molar basis 1.144 nM of 1 2 5I (MW 1 2 5I is 125) were reacted with 1.175 nM of GIP (MW is 5105) 1 25 Determined from the column prof i le , 78,9% of the I was reacted with the GIP or 0.902 nM/1.175 nM of GIP The specific act iv i ty of the GIP was therefore: 0.902 nM x 125 (MW) = 112.8 ng of 1 2 5I 112.8 ng/6 meg GIP or 112.8/72 = 1 .57 mCi/6 meg GIP or 262 jjCi/mcg GIP This calculation was representative of one column prof i le and had to be estimated for each new label l ing. To determine the quantity of "^I -GIP actually added to the assay the cpm (counts per minute) are converted to dpm (disintegration per minute). dpm counter cFulifil^"Wn^ien"cy x '100'%' dpm 14,700 4 4 1 yuCi = 2 , 2 2 x TO6 dps ( 1 Cu r ie = 3 , 7 x 1 0 1 0 dps) Therefore 1 4 , 7 0 0 dpm = 1 . 4 7 x 1 0 J = 6 . 6 2 x 1 0 " 3 « C i S ince there are 2 6 2 /iCi/mcg GIP 6 . 6 2 x 1 0 - 3 tiCi represents 6 . 6 2 x 1 0 " 3 = 0 , 0 2 5 x 1 0 " 3 meg o f GIP or 2 5 pg o f 1 2 5 I - G I P With the a d d i t i o n o f 4 , 0 0 0 cpm to the a s s a y , 4 / 1 0 of 2 5 or 1 0 pg o f 1 2 5 I - G I P was added. 45 RESULTS I Standard Curve Improvements The series of standard curves in Figures 15 through to 19 depict the increase in~sensitivity that occurred as a result of alterations in assay conditions, antibody production and antigen label l ing , The immprovements can be accounted for by alterations in a l l phases of the assay. The two most prominent changes are concerned with antibody and antigen. The use of conjugate for immunizing increased the antibody f inal di lution from 1/500 to 1/10,000. Changes in assay conditions and labell ing resulted in a further increase in usable di lution to 1/40,000. One interesting observation was the difference in sensit iv i ty be-tween standard curves in Figures 18 and 19. These assays ut i l i zed antigen with the same labell ing procedure and antibody. They differed only in respect to the new standard aliquots and antibody d i lu t ion , which yielded an increase in~sensitivity from 40 to 10 pg. This supports the importance of evaluating each new antibody in a sensit iv i ty assay involv-ing standard curves. II Specif ic ity of Antiserum Synthetic human gastrin (SH6-15 Leu), CCK-PZN, synthetic and natural porcine secretin, synthetic glucagon and motilin were tested for cross-reactivity with the antibody routinely used in the assay (Figure 20). A l l yielded B/F's within the standard deviation for zero GIP addition, thus indicating no cross-reactivity with the antibody, III Sensitivity,^Accuracy. aridi'Precision Each point and standard deviation of the curve in Figure 21 is I I I I _B F 1.00 -i GIP Standards in ng FIGURE FIFTEEN: Diluent Buffer: 1% human plasma, 1% Trasylol CO Antibody: 31/2/72 bleeding at a 1/500 final dilution 1 Labelling: 50 meg chloramine-T, mCi I, 5 meg GIP Separation: Charcoal-dextran (2.5 mg% charcoal) Sensit iv i ty : 500 pg GIP HIT Standard in ng FIGURE SIXTEEN: Diluent Buffer: 10% human plasma Antibody: 13/6/72 bleeding at a 1/6,000 f inal di lut ion Labelling: 50 meg chloramine-T, 2 mCi ^ 5 j 8 5 m C g gjp Separation: charcoal-dextran (1.25 mg% charcoal) with overnight mixing FIGURE SEVENTEEN: Diluent Buffer: same Antibody: 13/6/72 bleeding at a 1/10,000 final dilution Labelling: 40 meg chloramine-T, 2 mCi 1 2 5 I , 5 meg GIP Separation: same Sensit iv i ty : 60 pg FIGURE EIGHTEEN: Diluent Buffer: same Antibody: 24/7/72 bleeding et 1/10,000 f inal di lut ion Labelling: 40 meg chloramine-T, 2 mCi 1 2 5 I , 6 meg GIP Separation: same Sensit iv i ty : 40 pg so FIGURE NINETEEN: Diluent Buffer: same Antibody: 24/7/72 at a 1/20,000 f inal di lut ion Labelling: same Standards: new from mg weighing Separation: same Sensit iv i ty : 10 pg 51 o.eo -i Weight of Peptides (pg) ' I. FIGURE TWENTY: Evaluation of the c r o s s - s e n s i t i v i t y of secretin, glucagon, CCK-PZN and m o t i l i n to the GIP antibody. 53 based on seven determinations, The calculated sensit iv i ty with a P<or>0,01 is 25 pg by both Fisher modification and the Student t test, This indicated that this GIP assay was capable of determining unknown values down to the 25 pg level , The same curve replotted to include the protein interference correction of Nadeau and Zahnd (1971) is shown in Figure 22. GIP was added to charcoal treated plasma to y ie ld a concentration of 50 pg/100 jul . Ten determinations of 100 jul and 200 jul samples y ie ld -ed values of 54 + 4 (S.D..): and 100 + 10 (S,D,) respectively. This was an accuracy of 100%. The intra assay precision determined from nine repeats of normal pooled serum samples was 3,1%, An inter assay evaluation of precision based on ten repeat determinations of a 10% CCK-PZN standard was 11.9%, IV Plasma Dilution Assay A serum with high GIP content was assayed at various dilutions to determine i f the GIP in serum reacted the same as the GIP IV ut i l i zed for standard curves. Figure 23 indicates that the concentration of GIP in the plasma is dependent on the dilution at which the plasma is assay-ed. The values f i t ted to a standard curve, support the conclusion that the GIP in plasma is not identical in antibody reactivity to standard GIP (Figure 24). This data plotted as pg GIP for both standard and plasma on semi log paper indicates independence of plasma analysis to d i lut ion. This supports Yalow and Berson (1971b) contention that par-al lel ism of the log dose response curve is not as sensitive a measure of identical reactivity as the linear plot (Figure 23), 5* FIGURE TWENTY TWO: A replot of the data of Figure twenty one according to the method of Nadeau and Zahnd which corrects for protein interference. 1200 -i 0 0.1 0.2 0.3 0.4 ml of Serum /ml of Diluent Buffer FIGURE TWENTY THREE: Linear plot of the effect of plasma dilution on measured GIP content of a single serum sample di lut ion. FIGURE TWENTY FOUR: B/F of serum dilution f i t ted to a GIP standard curve to test for dilution effect 57 V Ten Percent CCK-PZN Assay Due to the origin of GIP from the side fraction of CCK-PZN, 10% CCK-PZN was evaluated for GIP content. It was also established as a quality control for individual assays. The parallel nature of the GIP IV and 10% CCK-PZN curves was indicated in Figure 25. Ten separate assay evaluations for GIP content of a 10% CCK-PZN preparation (batch number 26931) showed i t to contain 107 + 5.9 pg of GIP/1,000 pg. Lyophilized samples of this batch were prepared for use as a quality control for individual assays. The importance of re-testing new batches was indicated by the finding that batch number 297271 contained only 25 pg/1 ,000 pg of 10% CCK-PZN. On the basis of the ten assay values for 10% CCK-PZN (batch number 26931) a quality control chart was set up (Figure 26). The confidence l imits are + 2 S.D. of the mean value for 10% CCK-PZN reported as pg of GIP/ng of 10% CCK-PZN. The 10% CCK-PZN values obtained from subsequent assays are indicated in Figure 26. These values would be placed on the chart immediately prior to reporting assay unknown results. Based on this control, assay results from assay 4,35, 10, 13, 14 could not be re-ported as the 10% CCK-PZN values do not f a l l within the confidence l imits set. Another value of the chart is in indicating consistent assay changes. Assays 2, 3, 4 and 5 show a consistent increase in control value. A re-evaluation of assay constituents at this point would yield a decreasing 125 potency of antibody or a decrease in quality of I-GIP. I VI Normal Fasting GIP Serum Values and Ranges The normal fasting GIP level based on 45 determinations was F X x GIP Stondord o.oo -| , , . I 100 1000 10000 GIP Standards 8 10% CCK - PZN in pg FIGURE THWENTY FIVE: A comparison of the reactivity of GIP standards and 10% CCK-PZN with GIP antibody. 180 -| o o O cn c \ Cf> Q. c c o c o c o O 160 H 140 —I T « OvJ • ^ +1 6^ If) CD 120 A IOO H 8 0 A 6 0 H 4 0 - 1 X X X X n r 6 9 Assays 12 15 FIGURE TWENTY SIX: A quality control chart based on ten determinations of GIP content of a 10% CCK-PZN standard with 10% CCK-PZN standard v values of subsequent assay plotted. 60 163 +17 (mean + S .E . ) . The normal level had been decreasing since November 1972 at which time i t was 636 +31 (S.E.) . This is consistent with other radioimmunoassays (Yalow and Berson, 1972) and accompanies improved assay sensi t iv i ty . The normal fasting range for human GIP was established by the method of Hoffman (1971) as indicated in Table 4. As determined from the normal probability graph (Figure 27) the normal range was 0-300 pg/ml. VII Post Prandial Release of GIP Fasting serum levels were obtained from six subjects. Following a meal consisting of 4 oz. of orange ju ice , 9 oz, of whole milk, bacon, two eggs with hash-brown potatoes, toast with conserves, and coffee or tea; serum samples were taken every one half hour for a four hour period. Figure 28 shows the average serum GIP levels of the six sibjects +_ the S.E. Table 5 indicates the responses of the individual subjects. VIII GIP Serum Levels With Oral Glucose Tolerance Tests Serum samples were obtained following the oral administration of 75 mg of glucose to fasting normals. The GIP responses of five subjects are depicted in Table 6 and Figure 29. GIP levels following an oral glucose tolerance test in one ind i -vidual with insulin dependent diabetes is also indicated in Figure 29. T A B L E FOUR NO. OF CUM CUM S C A L E T E S T S SUM % 2 5 2 2 4.4 5 0 2 4 8.9 7 5 3 7 1 5 . 6 1 0 0 7 1 4 n i 1 2 5 4 18 4 0 . 0 1 5 0 5 23 5 1 . 1 175 1 2 4 5 3 . 3 2 0 0 4 28 6 2 . 2 2 2 5 4 32 7 1 . 1 2 5 0 5 37 8 2 . 2 2 7 5 4 4 1 9 1 a 3 0 0 4 4 5 1 0 0 . 0 100 200 300 400 GIP Fasting Serum Levels ( pg / ml) FIGURE TWENTY SEVEN: Probability graph plot of the frequency distribution of S GIP normal fasting values used to determine the GIP normal range. 1400 - i 285 FIGURE TWENTY EIGHT: Post prandial release of GIP. TABLE FIVE GIP SERUM LEVELS IN "SIX INDIVIDUALS FOLLOWING BREAKFAST TIME IN MINUTES FASTING 45 75 "" 105 135 165 195 225 255 SUBJECTS" 01 250 1050 1500 1250 1650 1850 1250 1600 775 02 150 1550 1515 1350 1400 1100 900 1150 1100 03 90 1375 1325 1600 1600 1550 1075 875 875 04 215 1325 1500. 950 1260 1350 1350 500 525 05 •  180 575 650 600 950 850 650 625 600 06 73 1250 1325 1250 875 825 1125 550 725 MEAN ' 142 1215 1263 1150 1217 1135 1020 740 765 S.D. 59 374 354 385 304 315 261 270 229 S.E. 26 167 158 172 136 140 117 121 102 168 1382 1421 978 1353 1275 1137 861 867 116 1048 1105 1322 1081 995 903 619 663 TABLE SIX GIP SERUM LEVELS DURING ORAL GLUCOSE TOLERANCE TESTS SUBJECT FASTING 5 MIN. 15 MIN. 30 MIN. 60 MIN. 120 MIN. 130 MIN. LEVEL 0.1 230 350 1500 1250 500 400 380 0.2 260 640 1340 1650 1230 1000 620 0.3 210 580 1760 1260 1160 660 520 0.4 280 375 700 750 750 350 300 0.5 350 450 750 1200 650 720 420 MEAN 266 478 1210 1222 858 626 448 S.D. 54 128 467 319 321 263 124 S.E. 24 57 209 142 143 117 55 CTl A 1 1 I 1 1 1 1 1 20 60 100 140 180 Fasting Level T ' m e M i n u t e s a f t e r Glucose Ingestion FIGURE TWENTY NINE: Serum GIP levels during oral glucose tolerance tests 6:7 DISCUSSION Gastric inhibitory polypeptide (GIP) was isolated from a side frac-tion of CCK-PZN (Brown, Pederson, Jorpes and Mutt, 1969), purified (Brown, Mutt and Pederson, 1970), and sequenced (Brown, 1971; Brown and Dryburgh, 1971). The pure material was found to inhibit gastrin, penta-gastrin, histamine and insulin hypoglycemia-induced acid and pepsin secretion (Pederson and Brwon, 1972). The inhibit ion of histamine stim-ulated acid secretion is particularly relevant, GIP is the only puri -fied polypeptide shown to have this effect. The inhibit ion is similar in magnitude to that obtained with fat in the duodenum, This is an important requirement for the enterogastrone or iginal ly defined by Kosaka and Lim (1930a). Purified GIP was also found by Barbezat and Grossman (1971) to be a potent stimulant of jejunal and i l ea l secretion. This property could be relevant to the eteology of increased small bowel secretion found with vibrio cholera and pancreatic cholera, In order to further evaluate GIP's physiological and pathological roles, a means had to be developed for measuring circulating GIP serum levels. Assay demands were based on known properties of other poly-peptide hormones. Low fasting levels required a very sensitive assay, Large increases in concentration of relat ively short duration must be followed accurately demanding a high degree of precision, The structur- . al s imi lar i t ies between GIP and other gastrointestinal polypeptides necessitates evaluation of the degree of cross reactivity between these peptides and the GIP antiserum. Gastrin radioimmunoassays have satisf ied a l l the above requirements and thus radioimmunoassay was the logical choice for GIP. 68 Radioimmunoassays are based upon the competitive inhibit ion of an antibody/labelled antigen reaction by unlabel led antigen, A dose re-sponse curve is obtained by challenging the labelled antigen-antibody complex with known amounts of unlabelled antigen, This standard curve is used as a reference for converting the displacement caused by un-known quantities of peptide in the serum, to absolute quantities, The assay requirements include an antibody that is specific for the peptide iof interest, a pure labelled peptide, and a standard, It is not necessary that standard and labelled peptide exhibit identical re-act iv i ty with the antibody, but i t is required that standard peptide and unknown peptide show identical immunoreactivity (Yalow and Berson, 1972), Since the immune reaction wi l l be affected by changes in the incubation environment, eg. pH, protein concentration and salt concentration, these must be constant for both standards and unknown (Yalow and Berson, 1971b), The theoretical aspects of radioimmunoassays have been presented at numerous symposia (Yalow and Berson, 1968c; Yalow and Berson, 1971a), Basically B/F = K(Ab° - b[H] ) where K is the equilibrium constant for antibody/antigen complex formation, Ab° is the molar concentration of combining sites on the antibody molecules at the di lution employed in the assay and b[H] is the fraction of total hormone concentration bound. From this relationship certain basic radioimmunoassay properties can be elucidated: 1) As b[H] increases the B/F decreases, 2) Since b[H] cannot exceed 1 or 100% binding, the detection of small changes in b[H] requires that Ab° is not too large. 3) b[H] consists of both that labelled and unlabelled [H], If a large amount of labelled [H] is added, i t w i l l l imit the amount of unlabel-69 led [H] detectable. 4) K is a measure of the energy of interaction of the antibody/antigen reaction and is a property of the antibody. As K increases, Ab° and b[H] can be decreased thus increasing the sensit iv i ty of the assay. An assay can be ut i l i zed for physiological and pathophysiological investigations following: 1) production of adequate antibody, labelled peptide and standard 2) optimizing Ab° and labelled [H] 3) standardizing conditions^ of incubation 4) Quantitative and qualitative evaluation of the assay Production of antibody with a high af f in i ty (avidity of antibody for antigen) and specif ic i ty was required for the GIP assay. Immuniza-tion of both guinea pigs and rabbits with GIP emulsified in Freunds Complete Adjuvant, yielded antisera of low af f in i ty and t i t e r (final dilution usable in the assay). Conjugation to bovine serum albumin resulted in a 5,000% increase in t i t e r (Figure 1). It can no longer be assumed that below a molecular weight of 5,000, proteins are not ant i -genic (Yalow and Berson, 1971a), This does not mean, however, that conjugation wi l l not be required. The increase in response to GIP following conjugation indicated that i t was necessary for this polypep-tide although i ts molecular weight is 5105. Polypeptide antigenicity is predominantly a function of species variation in structure (Odell et a l , T971). It follows that the larger the polypeptide the greater the .1ikelihood of alterations in amino acid composition amongst species. The GIP used for immunization was of porcine or igin. The lack of production of adequate antibody in rabbits 70 as compared to guinea pigs could be due to a greater s imi lar i ty between porcine and rabbit GIP, than between porcine and quinea pig GIP. Limited quantities of GIP excluded the use of a large series of animals for antibody response evaluation. Thus age and breeding could also have limited the GIP antisera production. If the animals were not immunologically competent (5.5 to 8.5 months for rabbits and 55 to 70 days for guinea pigs), injection could result in tolerance (Hurn and Landon, 1971). Also since the immune response is inherited, random breeding is essential when u t i l i z ing a small number of animals to i n -crease the probability of obtaining a good antisera (Yalow and Berson, 1971a). With these l imitations in mind rabbits cannot be ruled out as unsuitable for GIP antisera production. There appears to be confusion in the f i e ld as to whether pure or impure preparations of substances should be used for immunization. Both were used for GIP antibody production. Yalow and Berson (1971a) ind i -cated that crude preparations may yield higher a f f in i t y antisera than pure. This was based upon practical experience. Parker (1971) suggests that as long as pure peptide is available for label l ing , an impure material can be used for immunizing. To improve sensit iv i ty of the as-say, however, a pure material should be used. This is explained on the basis of heterogeneity of antibody response. A l l antibody in a single antiserum wi l l not have equal a f f in i t y for the antigen. As the amount of antibody of low af f in i ty increases the concentration of antibody re-quired in the assay also increases, thus resulting in a decrease in sensi t iv i ty . Crude preparations tend to increase the production of low a f f in i t y antibody. 71 The effect of the dose of antigen used for immunization can be ex-plained on the basis of high and low af f in i ty antibody production. Parker indicated that a decrease in antigen concentration stimulated production of high af f in i ty antibody while the converse was true for low af f in i ty antibody. This would explain an increase in t i t e r seen 20 days post immunization; since at that time the concentration in the animal would be much lower than immediately following immunization, The quality of the labell ing is important in determining the spec i f ic i ty , sensit iv i ty and precision of a radioimmunoassay (Hunter, 1969). A high concentration of iodinated molecular fragments and con-taminants w i l l increase the amount of labelled material that must be added to an assay, assuming that these do not interact with the antibody. If they do contain the immunologically competent part of the molecule they wi l l combine with the antibody but at a different K value than the intact polypeptide. Either condition would yield decreased assay sensi t iv i ty . GIP was a d i f f i c u l t peptide to label due to i t s sensit iv i ty to chloramine-T. Fragments produced were a particular problem during the early developmental stages. Recent evidence (Greenwood, 1971) indicat-ed that the addition of plasma in the eluting buffer used during the iodination pur i f icat ion, selectively adsorbs small peptide fragments. This is one variable that was not investigated during development. But i f this were the case, then an increase in the human plasma concentra-tion in the eluting buffer should increase the purity of the labelled product. Greenwood also suggests that saturation should be attempted with different proteins such as human serum albumin, bovine serum 72 albumin and globulins, to determine which is the most eff ic ient at re-moving labelled fragments. Early in the development of the assay i t was found necessary to perform label l ing in a si l iconized tube to prevent adsorption of mate-r ia l to the glass (Yalow and Berson, 1968a). Iodinated fragments and unreacted 125j a r e n o t adsorbed while intact hormone i s . This would result in a reduced concentration of labelled peptide in solution, mak-ing purif ication more d i f f i c u l t . The degree of 125j substitution in a molecule must be evaluated by comparing the improved assay sensit iv i ty with the increased probability of decay catastrophe found following high specific act iv i ty iodinations. Decay catastrophe is the radioactive production of labelled fragments following decay of one of the substituted iodines (in a d i - or t r i -iodinated preparation). GIP has two tyrosine molecules and thus is a possible candidate for decay catastrophe. At the present 125I/QIP ratio of 1:3, double iodination should be reduced to a minimum (Yalow and Berson, 1968a). The diluent buffer provides the environment for the antibody/anti-gen reaction and thus is extremely important, Although typical ly radio-immunoassays seem to be run at a pH of 7 to 8, the GIP assay was most sensitive at a pH of 6.5. This pH was f i r s t ut i l i zed because GIP's isoelectr ic point is approximately 7.5 and i t is an anionic protein. More d i f f i c u l t to determine than pH or ionic strength is the concen-tration of plasma and the amount of protection from the plasma required by the polypeptide. GIP was found to be very sensitive to protease act-i v i t y , a property shared with glucagon (Assan, et aj[, 1971) and para-73 c thyroid hormone (Arnaud et aT_, 1971) but not with gastrin (Yalow and Berson, 1971). Yalow and Berson (1971b) indicate various substances used to protect against protease act iv i ty . Trasylol was chosen for GIP. Trasylol is a competitive inhibitor of protease activation and a non-competitive inhibitor of protease act iv i ty (Dubber jet aj_, 1968). Below a Trasylol concentration of 250 KlU/ml of diluent buffer, GIP was not protected adequately from protease act iv i ty . This was reflected in a decrease in sensit iv i ty and an increase in the damage component of the assay (tubes containing only GIP and diluent buffer). Above that concentration there was a suppression of the standard curve (Figure 6). Although other individuals use Trasylol at double the concentration se-lected for GIP (Assan et al_, 1971 and Arnaud et al_, 1971), no reference in the l i terature could be found of a suppressive effect of Trasylol at high concentrations. Since the antibody/antigen reaction is not affect-ed by Trasylol (Forster, 1969), the suppression must be due to a direct action on the GIP molecule. Plasma added to diluent buffer serves two purposes; to eliminate adsorption to glass and to maintain a constant assay environment. Ideal-ly the only difference between the standard and the unknown, should be the concentration of the actual peptide being measured. In practice, however, the hormone is usually assayed in samples containing serum pro-teins which may introduce an effect of non-specific competitive binding. Many methods have been proposed to correct for this interference at the assay and/or analysis stage. Yalow and Berson (1971b) stress the im-portance of evaluating plasma dilutions thus providing an index of the susceptibi l i ty of an assay to protein interference. Corrections at the 74 assay level are numerous, Arnaud et (1971) routinely analyze their samples at three di lut ions, discarding samples that fai led to duplicate within preestablished l imi ts , They also equalized the concentration of plasma in the standard with that in the unknown tubes by the addition of hormone devoid plasma. Protein interference can also be reduced by adsorbing the antibody onto the incubate tube (Thorell, 1968), Nadeau and Zahnd (1971) correct for interference by incubating each different plasma evaluated with excess antibody. They reason that non specific competitive binding w i l l depend on the amount of protein present. Thus corrections based on incubating antigen and unknown alone w i l l y ield falsely high values when compared with the true assay situation in which antibody is also present. The use of an excess antibody tube in conjunction with the damage tube allows for a more complete evaluation of the extent of non specific interference. The GIP assay was developed to minimize the protein interference and also to correct for any interference that was s t i l l present. Ten percent human plasma was added to the buffer to minimize the plasma con-centration difference between unknowns and standards. The plasma had undergone prior charcoal treatment to remove any polypeptides. It was outdated plasma from the blood bank and thus should be devoid of pro-tease act iv i ty . Evaluation of protein interference was or iginal ly via the damage tube, but an excess antibody correction was incorporated. Since the conditions can never be total ly equalized throughout, the GIP assay was set up to incorporate reasonable control for protein interference. The method of Arnaud -et aj[, (1971), as previously men-tioned, is certainly closest to the ideal but i t would be total ly impractical for evaluating the large number of samples required during feeding studies involving numerous sequential determinations. Although an incubation time was established as optimum for the assay this was based on one particular antibody. The af f in i ty or association constant (K) is a function of the antibody and thus with changes in antibody af f in i ty there w i l l be consequent changes in opt i -mum incubation times. Each new antiserum must then require reevaluation. Incubations were carried out at 4°C because K is greatest at the lower temperature. * GIP antibody is used at high dilutions which necessitate longer incubation times. This approach is supported on theoretical grounds by Parker (1971). He states that during long incubations the antigen wi l l redistribute i t s e l f to high af f in i ty antibody, This reduces the amount of low af f in i ty antibody/antigen complexes which more readily disas-sociate during separation. A decrease in disassociation wi l l y ie ld an increase in assay sensi t iv i ty . The antibody/antigen complexes do not spontaneously precipitate because of the small quantities of antibody and antigen employed in radioimmunoassays. As a result some means must be adopted for pre-c ip i tat ing either the free antigen or antibody/antigen complexes, Daughaday and Jacobs (1971) described six methods of separating antibody/ antigen complexes from free antigen: 1) electrophoretic and chromatoelectrophoretic methods 2) gel f i l t r a t i o n 3) nonspecific precipitation of hormone-protein complexes 4) immunoprecipitation of soluble hormone-protein complexes 76 5) solid-phase adsorption of hormone 6) solid-phase adsorption of antibody The choice of method for a particular assay must involve considera-tion of completeness of separation, reproducibil ity, f l e x i b i l i t y of con-ditions and general convenience with respect to time and effort . Meth-ods 2 to 5 inclusive were evaluated for use with the GIP assay. Dioxane, double antibody and charcoal-dextran were found to provide a degree of separation. Charcoal-dextran was the method of choice. The concentra-tion of charcoal-dextran presently employed was found to y ie ld optimum exclusion of antibody/antigen complex and relative lack of interference from nonspecific proteins in the incubation volume (Figure 10 and 11), Coating the charcoal with dextran was i n i t i a l l y developed by Herbert (1968) to improve exclusion of the antibody/antigen complex. Palmieri, Yalow and Berson (1971) and Raptis (1971) found no increase in effectiveness of separation with charcoal-dextran as opposed to char-coal alone. GIP separation was improved with dextran coating, support-ing the findings of Herbert (Lau, Gottliie and Herbert, 1966), The use of coating is l ike ly dependent upon the particular peptide assay system, Herbert (1968) indicates that the charcoal-dextran separation is almost instantaneous. The half hour time delay between addition and centrifugation in the GIP assay allows for some charcoal precipitation prior to centrifugation. The major reason for this incubation period is to allow for standardization of separation time. If centrifugation was accomplished immediately following addition, the time delay would vary with the size of the-assay. The charcoal-dextran technique was a rapid and simple method of 77 separation, both important considerations for a routine c l in i ca l test , It was also considerably more economical than the double antibody meth-od, and as compared to methods one and s ix , required very basic equip-ment. Charcoal-dextran had therefore satisf ied the c r i te r ia previously mentioned by Yalow and Berson for separation in radioimmunoassays. It provided the most complete separation (Figure 10), was reproducible, was very convenient and reasonably f lex ib le . Once the techniques had been established, the assay had to be eval-uated quantitatively and qual i tat ively . Quantitation involves deter-minations of spec i f i c i t y , sens i t i v i ty , accuracy and precision. A qualitative appraisal comprises determination of plasma effect by measuring samples at different dilutions (Yalow and Berson, 1971b). The extreme specif ic i ty of the antibody was determined by compar-ing a GIP standard curve to the curves obtained with gastrin, CCK-PZN, secretin, glucagon,and moti l in . At levels up to 10 ng there was no zr cross reactivity of any of.the peptides with the GIP antibody (Figure 20). As mentioned earl ier this was extremely important in view of the structural s imi lar i t ies between GIP, glucagon and secretin. Equally important is the origin of GIP, glucagon, secretin, CCK-PZN and motilin from the endodermal tissues of the fore- and mid-gut regions. The l i k e -lihood of a l l these substances being released simultaneously is high and thus many sera measured for GIP content would have a high gut peptide content. The specif ic i ty exhibited by the antibody provides assurance that the competitive binding exhibited by unknown samples i s , in fact , a measure of GIP concentration. The sensit iv i ty as measured by the lowest level of standard s igni f -78 icantly different from zero competition was 25 pg. The particular curve (Figure 22) was not as sensitive (evaluated visually as the slope of the curve) as compared with the results presented in Figure 19, This was due to the increasing age of the labelled antigen which was nearing the three week expiry date. Twenty five picograms was thus the minimum sensit iv i ty expected with the GIP assay and therefore 25 pg/100 /jl or 250 pg/ml was the minimum routinely readable serum level . Replotting the data by the method of Nadeau and Zahnd (1971) yielded the curve indicated in Figure 22. This was preliminary data as the meth-od requires a 1/200 dilution of antibody to be tested with each separate plasma. Since normal di lut ion is 1/40,000, a large stock of antibody would be required before this could be evaluated with a large number of samples. A comparison of the sensi t iv i t ies of curves in Figures 21 and 22 based on slope would indicate a marked improvement in Figure 22 due to the protein interference correction. Feldman and Rodbard (1971) have indicated an additional c r i te r ia of sensit iv i ty . The slope should be steep in the area of zero competition and yield a high intercept. Comp-aring Figures 21 and 22 again indicates a much steeper slope in Figure 22 thus yielding larger changes between different ordinate values, This would allow for easier differentiation between unknown values within this area of the curve. Neither curve sat isf ies the c r i te r ia for a high intercept. Figure 19, which represents data from an assay u t i l i z ing newer antigen, comes much closer to satisfying this c r i te r ia , Improvements in standard curve sensit iv i ty represented in Figures 15 to 19 are due mainly to improvements in labell ing of antigen and antibody production. As the labell ing procedure was standardized, 79 further assay sensit iv i ty increase would require improved antibody a f f in i t y . This could be achieved by immunizing a new series of animals as indicated previously. Accuracy as determined by measuring plasma to which a known amount of GIP had been added was 100%. This indicates that the assay was capable of measuring a l l the free GIP present in serum unknowns. Precision of intra assay measurements were a l l under the 10% maximum acceptable l i m i t , and usually under 5%. Inter assay precision as evaluated by repeat 10% CCK-PZN measurements was 11.9%. These deter-minations were made during stages of assay improvement, and thus deviat-ed more than would be expected with assay s t a b i l i t y . The precision of the assay was s t i l l considered adequate for unknown evaluations. Qualitative validation of the assay was assessed by the method of Yalow and Berson (1971b). The results depicted in Figures 23 and 24 did not indicate identical reactiv i ty of the GIP standard and unknown. Yalow and Berson mention two possible reasons for th is : differences in hormonal cross-reactivity factors yield plasma di lut ion values that parallel the standard curve. This indicates decreased competitive binding similar to the curve obtained with 10% CCK (Figure 25). Since the GIP dilutions resulted in inconsistant reactivity (a decrease with lower dilutions) i t must have been due to non specific binding effects. Of the non specific factors indicated by Yalow and Berson (L971b), ionic environment, pH, presence of heparin and dif ferent ial effect of protein; only the effect of serum proteins could account for an apparent decrease in concentration with decreased d i lut ion. This decrease is measured as an increase in the B/F ra t io ; i e , Serum proteins (albumin 80 and gamma globulin) w i l l competitively inhibit the antibody/antigen reaction (Hunter,1968); yielding a decrease in antibody/antigen formation. There w i l l be simultaneous formation of albumin/antigen complexes that w i l l precipitate with the antibody yielding a false B elevation. This can be corrected by an excess antibody evaluation (Yalow and Berson, 1971a). Alternatively, the serum to be investigated could be rendered peptide-free by extraction with charcoal and added to the standard curve. This is impractical in routine assays. Figure 25 indicates another qualitative validation of the assay apart from serum di lut ion . The parallel nature of the curve, implies competitive binding of the antibody with GIP in both standards and 10% CCK-PZN. This validates the ab i l i t y of the assay to measure the GIP content of tissue samples. A l l the quantitative evaluations of the GIP radiommunoassay sup-ported i ts use for serum analysis. The qualitative plasma di lut ion did indicate a problem with competitive protein binding, however, methods for controlling and/or correcting i t were suggested. More important was the fact that the test was done only once. The results should be re-peated on a greater number of samples before definite conclusions are made. Prior to further evaluations the unknown could be analyzed rou-t inely at multiple di lut ions. Once these parameters had been determined and the assay accepted as suitable for unknown measurements, a quality control procedure had to be established. A means had to be developed for evaluating each new assay prior to reporting results. To accomplish this 1 ng samples of a 81 single batch of 10% CCK-PZN were assayed in a series of experiments, The mean and standard deviation of the repeat values were calculated and a chart was set up indicating the mean and establishing boundary lines at +_ 2 S.D. Each subsequent assay included a 10% CCK-PZN measure-ment which was plotted on the graph. Any values fa l l ing outside the standard deviation indicated a possible assay fa i lure , If two succes-sive assays yielded 10% CCK-PZN values outside of the boundary lines the unknowns in those assays must be reevaluated prior to reporting. Two successive assays are necessary because 1 in 20 assays would be ex-pected to y ie ld "out of bounds" 10% CCK-PZN values due to chance alone (+2 S.D. yields a 95% confidence level ) . The chart can supply information on shifts in procedures as well as information on individual assays. A shi f t occurred between assays 1 to 5, and is indicated by the gradual increase in 10% CCK-PZN values during this period. Shifts can be due to many causes, Particularly suspect with radioimmunoassays would be antibody af f in i ty change or labell ing efficiency change. Once a shift l ike this was noted the tech-nique must be reevaluated until the particular cause was discovered. Shifts were d i f f i c u l t to determine i f the results were not available for .visual inspection. The values indicated on the chart- in Figure 26 were obtained during a period of assay improvements and thus the results were not as consistent as one would expect from a stable assay, Once the assay is fu l ly established further controls with control charts should be incorporated in each assay, Included among these should be two patients pooled serum; one a pooled normal and the other a pooled abnormally high.- These three samples would evaluate the assays' accur-82 acy (10% CCK-PZN), precision (10% CCK-PZN and normal patient pool) and ab i l i t y to determine abnormal sera (abnormal high pool), Thus the 10% CCK-PZN was an in i t ia t ion of an established quality control program for the assay. The normal range was evaluated by the method of Hoffman (1971), The basis of the calculations involves the assumption that a large num-ber of determinations of serum GIP levels of different individuals should yield a normal distr ibut ion. Thus a plot of the percentage of values fa l l ing within a frequency distribution should yield a sigmoid curve. The probability graph paper is a log transformation to convert the sigmoid curve into a straight l ine . If a straight l ine is obtained by plotting a l l the values, with the exclusion of the obviously abnormal, the data can be considered normally distributed and the normal range can be read directly from the probability plot as + 2 S,D,, The estimate was based on 45 determinations of the serum fasting level of GIP from patients without gastro-intestinal disorders, A straight l ine was obtain-ed and thus the normal range was determined to be +_ 2 S,D,, The range should be re-evaluated on a larger sampling (150 samples) when the data is available. This is particularly important for GIP as the normal value decreased considerably from November to February, A further reduction would be expected from the observations with the gas-t r in radioimmunoassay (Yalow and Berson, 1972), as indicated ear l ier , This would follow increased assay sensit iv i ty resulting from improve-ments in any of the assay stages, particularly increased antibody a f f i n -i t y . Normal ranges are tradit ional ly taken as + 2 S.D, because the i n -83 dividual determinations are affected by a large number of extraneous fac-tors, Included among these factors are: sex, age, exercise, d iet , emo-t ion , posture, tourniquet techniques, drugs and f lu id intake (Hoffman, 1971). Age has been found to be a particularly important source of var-iation with other gastro-intestinal polypeptides, yielding increased fasting levels with age (Yalow and Berson, 1972), With these l imi ta -tions in mind the normal range is necessary for c l in ica l evaluation to provide guidelines for distinguishing between normal and abnormal results. Following determinations of the normal range and establishment of at least one control for evaluating individual assays, preliminary i n -vestigations were undertaken to evaluate GIP serum levels during feeding studies, glucose tolerance tests and in certain pathological states, None of the studies attempted to accurately quantitate responses as the number of subjects included was too small. Results and conclusions were therefore of a preliminary nature. The intent was one of sampling the areas available for study following development of the assay, The feeding study provided evidence of GIP release following food intake. No specific secretagogues could be determined from the mixed meal ingested. It does support the position of GIP as a human gastro-intestinal peptide, released into the blood stream by a feeding stimulus, This is part proof of GIP's hormonal status, Final hormonal proof would require measurement of a concomittant decrease in acid and pepsin secretion following increased serum levels of GIP, A study of GIP release following ingestion of a hypertonic solution was provided by the glucose tolerance tests (Figure 29), Not only did the results reveal the stimulatory effect of hypertonic solutions with 84 respect to GIP but also the possible insulin secretagogue act iv i ty of GIP i t s e l f . Involvement of a gastro-intestinal polypeptide in insulin release has long been suspected due to the increased insulin response with oral as opposed to IV glucose administration (Mclntyre et_ al_, 1965), Attempts to determine which polypeptide was responsible have to date been inconclusive (Rehfeld, 1972, and Yalow and Berson, 1972), None of the gastro-intestinal polypeptides (gastrin, CCK-PZN, secretin and gut glucagon) tested have shown increased insulin secretion in either invtvo or invitro studies. Dupre (personal communication to J . C. Brown) studied the effect of pure CCK-PZN, 10% CCK-PZN and GIP on the invitro release of insulin from pancreatic s l ices . Pure CCK-PZN did not stimulate insulin release while 10% CCK-PZN did. Pure GIP was found to be a potent stimulator of insulin release. The assumption from this was, of course, that the action of 10% CCK-PZN was due to GIP contamination. Following these re-sults invivo studies were in i t ia ted . Subjects were glucose loaded to y ie ld a blood level of 160 mg% at which time serum insulin levels were 40-50 uU/ml. An intravenous infusion of 1.0 meg of GIP/min was continu-ed for 30 minutes and yielded insulin levels up to 100 uU/ml. With the development of the radioimmunoassay i t was possible to measure GIP levels following oral glucose tolerance tests (Figure 29), The data indicated a GIP response similar in temporal sequence to the blood glucose increase. Cataland (personal communication) has measured serum GIP, glucose and insulin levels following glucose ingestion. The peak GIP response occurred at 15 to 30 minutes (Figure 29) while the insulin and glucose peaks were at 30 minutes. Also included in Figure 29 85 is a total lack of GIP response to oral glucose in a patient who was insulin dependent. Al l the data supporting GIP as an insulin secreta-gogue were of a preliminary nature. Final support awaits further i n -vestigation but the positive findings with both invivo and invitro X - ^ .r. tests were certainly more promising than that found with the other gastro-intestinal polypeptides. Cellular localization of GIP was recently reported using the ant i -body supplied from this laboratory and presently used in the radio-immunoassay (Polak et al_, 1973). The cel ls were of the APUD (gastroin-testinal endocrine polypeptide) type and found predominantly in the mid-zone of the duodenum, and to a lesser extent in the jejunum. The areas of high GIP immunofluorescence corresponded best with the Dl endocrine cel l of the small bowel. The areas of GIP immunofluorescence were d is -t inct from the S cel l (secretin) and the L cel l (enteroglucaggn) and considerably more abundant than the S c e l l . Considering GIP's known role as an exogenous inhibitor of acid secretion i t was logical for GIP to be implicated in disorders with high and low acid secretion. Duodenal ulcers are usually associated with high acid secretion. Polak et_ al_ (1973) were unable to identify GIP cel ls in patients with duodenal ulceration. Decreased or absent gastric acidity is a symptom of pernicious anemia and gastric carcinoma. Biopsies of small bowel from patients with gastric carcinoma were exam-ined by Polak et a^ (1973) and found to have increased GIP immuno-fluorescence. Preliminary data on two pernicious anemia cases yielded serum GIP levels four times greater than normal fasting levels . These data were extremely tentative due to the small number of cases examined 86 but they did support the possibi l i ty that GIP may be involved in path-ological conditions associated with alterations in gastric acidity . Cause and effect w i l l have to be investigated thoroughly but i t is reasonable to consider that GIP may be valuable as a therapeutic agent in cases of duodenal ulceration to reduce gastric acidity . GIP's role as a stimulator of intestinal secretion has implicated i t in disorders associated with increased intestinal secretion. Two important examples are vibrio cholera and pancreatic cholera. Both conditions are associated with profuse watery diarrhea yielding decreas-ed serum potassium and acid base inbalances, Pancreatic cholera is due to an endocrine secreting pancreatic tumor that has been identified by immunofluorescence to contain GIP or a GIP l ike material (Elias et a l , 1972). This condition is usualTy;iassocciated:3.wi:th: achlbrhydria?and alterations in the glucose tolerance curve. Both of these symptoms could be explained on the basis of known GIP actions mentioned previous-ly. Preliminary attempts to measure serum GIP levels in these patients, however, have been unsuccessful. Following the development of the radioimmunoassay for GIP, preliminary studies support GIP's position as a human circulating poly-peptide produced primarily in the ARUD cel ls of the duodenum. GIP serum levels increase in response to a feeding stimulus, indicating i t may be functionally important in the feeding response, Pathological conditions in which GIP may be involved include: duodenal ulceration, pernicious anemia, vibrio and pancreatic cholera. Much work wi l l be required in a l l of these areas in order to establish GIP's actions: physiological and pathological. 87 BIBLIOGRAPHY Al ley, A. and MacKenzie, D. W, Dissociation of the functional propert-ies of gastric glands under the influence of fat . Am. J . Dig. and Nut. 1^ 333 (1934-35). -Arnaud, C. D.,.Tsao, H. S . , and L i t t led ike , T, Radioimmunoassay of human parathyroid hormone in serum. J . C l in . Invest. 50, 21 (1971). Assan, R,, Tchobroutsky, G., and Derot, M, Glucagon radioimmunoassay: technical problems and recent data. .In Immunological Methods in Endocrinology, 83, Levine, R, and Pfe i f fer , E. J , , , E d . (Hormone and Metabolic Research, Suppl. 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